Fig 1.
The SPI protein interacts with DCP1.
(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His6 with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His6 (lower part) antibody staining. GST-SPI-PBW-His6 (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRETE (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see Materials and Methods). Mean FRETE’s for 35Spro:YFP-gSPI and 35Spro:DCP1-CFP (n = 11 cells) or 35Spro:YFP and 35Spro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (n = 31 stationary PBs derived from whole cell samples). Two-tailed student’s t test was performed to compare FRETE between 35Spro:YFP-gSPI/35Spro:DCP1-CFP and 35Spro:YFP/35Spro:DCP1-CFP for each group (*** p < 0.001). (E) Representative images of 35Spro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.
Fig 2.
SPI and DCP1 interact at P-bodies in BiFC assays.
Fusion proteins are expressed under the control of the 35S promoter. (A) Interaction of YFPN-SPI and YFPC-DCP1 (left) in transiently transformed N. benthamiana leaves 72 h post-transfection at DCP2-mCHERRY labeled P-bodies (middle). Right pictures show the overlay of the left (green) and middle (magenta) pictures. (B) Interaction of YFPN-SPI and YFPC-DCP1 in cytoplasmic dots (left) in N. benthamiana leaves coexpressing free RFP as transformation control (middle). Right pictures show the corresponding transmission picture. (C–F) Representative images of BiFC negative controls. No YFP signal in leaf epidermis cells coexpressing YFPN-SPI-PBW and YFPC-AtMYC1 in combination with DCP2-mCHERRY (C) and free RFP (D). No YFP signal in leaf epidermis cells coexpressing YFPC-DCP1 and VPS25-YFPN in combination with DCP2-mCHERRY (E) and free RFP (F). Scale bar: 50 μm.
Fig 3.
Colocalization studies with YFP-fusions of SPI (column I) and DCP2-mCHERRY-labeled P-bodies (column II) in transiently transfected Arabidopsis leaf epidermis cells.
Column III presents the overlay picture between columns I (green) and II (magenta). (A) Under nonstress conditions, YFP-gSPI (upper row) and YFP-SPI-PBW (lower row) are evenly distributed throughout the cytoplasm. (B) YFP-gSPI (upper row) and YFP-SPI-PBW (lower row) accumulate at DCP2-mCHERRY-labeled P-bodies after incubation of transfected leaves on ½Murashige and Skoog (MS) medium supplemented with 140 mM NaCl for 10 h. Scale bar: 25 μm.
Table 1.
P-body number in leaf epidermis cells transiently expressing DCP1-mCHERRY.
Average numbers of P-bodies are provided for Col-0, spi-2 and spi-4 under nonstress (½MS) and salt stress (½MS supplemented with 140mM NaCl for 10 h) conditions. Each biological replicate (n) comprises at least 30 cells. SD values represent standard deviations. Two-tailed student’s t tests were performed to compare stress and nonstress conditions.
Fig 4.
Blocking translation leads to a reduction of P-body number.
Changes of P-body number were analyzed in whole leaf areas of transgenic plants expressing DCP1-YFP. (A) Whole leaf areas are presented before (0 min) and after continuous CHX treatments (45 min and 90 min). Scale bar: 50 μm. (B) Reduction of P-body number (in %) relative to untreated samples (time point 0) determined 45 min and 90 min after CHX treatment. Data denote the average from seven biological replicates. SD values represent standard deviations. No statistical differences were found between wild-type and spi mutants (Two-tailed student’s t test).
Fig 5.
spi mutants display salt hypersensitivity.
Relative changes of root length after 10 d on ½MS plates supplemented with (A) 100 mM, 125 mM, and 150 mM NaCl or (B) 100 mM and 250 mM Mannitol (Man). Data in (A) and (B) were normalized to nonstress conditions and denote the average from three independent biological replicates (n = 12 seedlings each). Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare spi alleles and Col-0 exposed to the same conditions (* p < 0.05; ** p < 0.01; *** p < 0.001). (C) Cotyledon greening of seedlings measured after 14 d on ½MS plates supplemented with 150 mM NaCl. Greening efficiencies (in %) denote the average from three independent biological replicates (n = 12 seedlings each). Errors represent standard deviations. Two-tailed student’s t tests were performed to compare spi alleles and Col-0 exposed to the same conditions (* p < 0.05; ** p < 0.01; *** p < 0.001). (D) Diameter of leaf rosettes (in cm) of 32-day-old plants, measured after irrigation with ½MS only (control) or ½MS supplemented with increasing NaCl concentrations on every second day (two times ½MS + 50 mM NaCl and two times ½MS +100 mM NaCl). Data denote the average from three biological replicates (n = 14 plants each). Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare spi alleles and Col-0 exposed to the same conditions (* p < 0.05; ** p < 0.01; *** p < 0.001). (E) Representative images of 30-d-old Col-0 and spi-2 plants grown under nonstress (½MS) and salt stress conditions (irrigation two times with ½MS + 50 mM NaCl and one time with ½MS + 100 mM NaCl in alternation with ½MS every second day) on a sand–soil mixture. Scale bar: 1.5 cm.
Table 2.
Significantly changed gene expression between Col-0 and spi.
Data present only those with a q-value < 0.01, Benjamini Hochberg (related to Fig 6).
Fig 6.
(A) Mapman visualization of log2-fold changes in Col-0 upon salt treatment and (B) the corresponding 25 most strongly enriched GO terms. Darker colors in GO term categories represent higher q-values (BH-corrected). (C) Mapman visualization of log2-fold changes in spi upon salt stress induction and (D) the corresponding 25 most strongly enriched GO terms in spi. For color codes see 6C. (E) Venn diagram comparing the salt stress-dependent up-regulation of transcripts in Col-0 (wt) and spi (see also S8 Table).
Fig 7.
mRNA decay is not accelerated in spi mutants under nonstress conditions.
mRNA stabilities (in %) were determined 3 h and 6 h after application of Actinomycin D (ActD), relative to time point 0 under nonstress conditions for RD29B (A), TZF3 (B), CIPK9 (C), and ABI1 (D). Data denote the average from three independent biological and two technical replicates. Error bars represent the standard error of the mean. Two-tailed student’s t tests were performed to compare 18S rRNA normalized expression levels either of spi alleles with that of wild-type (* p < 0.05; ** p < 0.01 of all spi alleles), or of ActD-treated samples with that of untreated samples (time point 0) (° p < 0.05; °° p < 0.01 of all treated samples).
Fig 8.
Role of SPI in the regulation of mRNA stability.
mRNA stabilities (in %) were determined 3 h and 6 h after application of Actinomycin D (ActD) relative to time point 0 under salt stress conditions (200 mM NaCl in ½MS liquid medium for 4 h). mRNA decay was determined for RD29B (A), TZF3 (B), CIPK9 (C), and ABI1 (D). Data denote the average from three independent biological and two technical replicates. Error bars represent the standard error of the mean. Two-tailed student’s t tests were performed to compare 18S rRNA normalized expression levels either of spi alleles with that of wild-type (* p < 0.05; ** p < 0.01 of all spi alleles), or of ActD-treated samples with that of untreated samples (time point 0) (° p < 0.05; °° p < 0.01 of all treated samples).
Fig 9.
mRNAs of RD29B and TZF3 form cytoplasmic granules.
16BoxB-gRD29B (A) and 16BoxB-gTZF3 (B) are indirectly visualized by the LambdaN22-VENUS reporter in transiently transfected leaf epidermis cells (related to Tables 3 and 4). Column I presents the LambdaN22-mVENUS reporter, column II DCP1-mCHERRY, column III the overlay of column I (green) and II (magenta). Rectangular image magnifications present P-bodies nonoverlapping (white arrowheads) and P-bodies overlapping (yellow arrowheads) RNA accumulations. Scale bar: 25 μm.
Table 3.
mRNA granules localize to P-bodies (related to Fig 9).
mRNA accumulations overlap with P-bodies under nonstress (-NaCl) and salt stress (+NaCl) conditions. Data denote the average (in %) of 65 cells (RD29B) or 120 cells (TZF3).
Table 4.
Numbers of mRNA granules per cell (related to Fig 9).
Average number of RD29B (n = 65 cells) and TZF3 (n = 120 cells) mRNA granules under non stress (-NaCl) and salt stress (+NaCl) conditions. SD values represent standard deviations.
Fig 10.
Interspecific interactions between SPI-PBW and DCP1 homologs from mammals and yeast.
(A) Yeast two-hybrid interactions. Top part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interactions between the SPI-PBW N-terminally fused to the GAL4 Binding Domain (BD) and the human DCP1 isoforms (DCP1a and b) and yeast DCP1p, N-terminal fused to the GAL4 Activation Domain (AD) on selective dropout medium lacking leucine (-L), tryptophan (-W) and histidine (-H), supplemented with 3 mM 3-Aminotrizole (3AT). GFP N-terminal fused to the GAL4-AD has been included as negative control. (B) Coprecipitations of bacterially expressed proteins. GST-SPI-PBW-His6 (arrowhead, ~110 kDa) coprecipitated with MBP-DCP1a (~90 kDa), MBP-DCP1b (~92kDa) and MBP-DCP1p (~60 kDa), but not with MBP (~42 kDa) as negative control. Throughputs (TP), last wash fractions (WF), and resin bound fractions (B) are visualized by α-MBP (upper row) and α-His6 antibody staining (lower row). Samples detected on different blots are separated by lines.
Fig 11.
Inter- and intraspecific interactions between FAN-PB and DCP1 homologs.
(A) Yeast two-hybrid interactions. Top part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interactions between the FAN-PB N-terminally fused to the GAL4 Binding Domain (BD), the human DCP1 isoforms (DCP1a and b) and the Arabidopsis DCP1, N-terminally fused to the GAL4 Activation Domain (AD) on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 3 mM 3-Aminotrizole (3AT). GFP N-terminal fused to the GAL4-AD has been included as negative control. (B) Coprecipitations of bacterially expressed proteins. GST-FAN-PB (arrowhead, ~70 kDa) coprecipitated with MBP-DCP1a (~90 kDa), MBP-DCP1b (~92 kDa) and MBP-DCP1 from Arabidopsis, but not with MBP (~42 kDa) as negative control. Throughputs (TP), last wash fractions (WF), and resin bound fractions (B) are visualized by α-MBP (upper row) and α-GST antibody staining (lower row). Samples detected on different blots are separated by lines.